Advances in Safety Pharmacology: Utilizing ipsc-derived cardiomyocytes in early stage safety pharmacology investigations. Blake Anson, PhD.

Transcription

1 Advances in Safety Pharmacology: Utilizing ipsc-derived cardiomyocytes in early stage safety pharmacology investigations Blake Anson, PhD.

2 Presentation Outline I. Safety Pharmacology and in-vitro models Need for human cardiac model Potential for stem cell technology to provide a solution II. Functional characterization of stem cell derived cardiomyocytes Genomic, protein, metabolic, electrophysiological III. Methods for interrogating cardiomyocyte function Electrophysiology, Ca2+ handling, Contractility Direct and Indirect (screening) methodologies IV. Case studies Retrospective Analysis V. Summary Mechanistic and Phenotypic Approaches 2

3 Safety Pharmacology Key functional endpoints From M. Pugsley Current common in-vitro models include cell lines and non-human cellular preparations. 3

4 The Heart Beat: A Remarkable Feat! Your heart is an electrically driven pump. It usually beats times a minute, or about 100,000 times a day, or about 35 million times a year, or about 3 billion times in a normal life span. If the normal pumping rhythm is severely disrupted for more than a few minutes, irreversible multi-organ damage and death occur

5 ipsc-derived cardiomyocytes Utility Cardiotoxicity: Two basic mechanisms Drug induced Arrhythmias Torsades de Pointes (Hismanal ) Vorperian et al, JACC, 15: , Biochemical induced events o Cell Permeability o Cell energetics o Oxidative stress o Mitochondrial dysfunction o Necrosis o Apoptosis The two toxicities can be species specific and may not be causal or linked The ideal test system is species specific and biologically relevant. Human ipsc-derived cardiomyocytes 5 5 5

6 Why Human? Mammalian Heart Variability in Ventricular Action Potential Duration Larger mammals have slower rates Longer APD Greater endo to epi dispersion of repolarization 6 Guo et al, Heart Rhythm. 5: ,

7 Why Human? : Very Different Ionic Mechanisms Between Large Mammals and Rodents Human Mouse P P 7 Salama and London, J Physiol. 578:43-53,

8 8 Human Stem Cell Derived Cardiomyocytes Human cardiomyocytes ES or ipsc source material Recapitulate cardiac cellular behavior Unlimited Quantity High Purity Relevant Biology Broad End-use Platform Utility

9 Human Tissue Cells ips Cell vs. ESC Origin INDUCED PLURIPOTENT STEM CELLS NEURONAL CELLS HEMATOPOIETIC CELLS CARDIOMYOCYTES RENAL CELLS EMBRYONIC STEM CELLS HEPATOCYTES Source: CDI website;

10 Cell Purity Stem Cell-Derived Models - Requirements Quality, Quantity, Purity Key Characteristics for ipsc use in Safety Pharmacology Purity Highly pure cells yield accurate results Quantity Must be able to support large scale investigations Quality Exhibit key cellular characteristics Recapitulate normal human biology Reproducible Known and relevant genotype Target Cell (non proliferating) Non-Target Cell (proliferating) Days in Culture

11 Presentation Outline I. Safety Pharmacology and in-vitro models Need for human cardiac model Potential for stem cell technology to provide a solution II. Functional characterization of stem cell derived cardiomyocytes Genomic, protein, metabolic, electrophysiological III. Methods for interrogating cardiomyocyte function Electrophysiology, Ca2+ handling, Contractility Direct and Indirect (screening) methodologies IV. Case studies Retrospective Analysis V. Summary Mechanistic and Phenotypic Approaches

12 12 hipsc-derived Cardiomyocytes Genomic and Transcript Characterization Comparative Analyses whole genome profiling ~200 Cardiac genes were identified from the Novartis GNF expression atlas Expression levels of these genes were obtained from adult human heart mrna library (ambion) Expression levels of these genes were obtained from pure populations of ipsccardiomyocytes over a 3 month period (d028 to d120) Relative levels compared with each other Purified ipsc-cardiomyocytes (ipsc-cardiomyocytes) show a stable cardiac expression profile that matches adult human tissue Adapted from Babiarz et al., 2011

13 13 Human ipsc Cardiomyocytes Protein Expression From Kattman et al., 2011

14 14 14 Human ipsc Cardiomyocytes Metabolic Characterization ipsc-cardiomyocytes utilize non-glycolytic substrates Adapted from Rana et al., 2012 ipsc-cardiomyocyte mitochondria are functional ipsc-cardiomyocytes utilize mitochondrial oxidative phosphorylation

15 15 Human ipsc-cardiomyocytes Contractility / Ca 2+ handling Characterization Contraction Sunny Z. Sun Determined by edge detection Ca 2+ Transients

16 Human ipsc-cardiomyocytes Depolarizing Currents 16 I Na I Ca-L Ma, et al, Am. J. Physiol., 2011

17 Human ipsc-cardiomyocytes Repolarizing Currents 17 I Kr I Ks Ma, et al, Am. J. Physiol., 2011

18 ipsc-cardiomyocytes Other Currents 18 I funny I K1 I to Ma, et al, Am. J. Physiol., 2011

19 Frequency ipsc-cardiomyocytes GPCR Pathways 19 Spontaneous Action Potential Beating Rate (MEA): G as b1 Isoproterenol G aq a1 Phenylephrine G ai m2 Carbachol Concentration (mm) Control Drug ipsc Cardiomyocytes have the appropriate GPCR pathways

20 Stem Cell -Cardiomyocytes Action Potentials ipsc-cms Ma, et al, Am. J. Physiol., 2011 hes-cms Peng et al., 2010 Stem cell derived CMs exhibit cardiac like action potentials 20

21 21 21 Presentation Outline I. Safety Pharmacology and in-vitro models Need for human cardiac model Potential for stem cell technology to provide a solution II. Functional characterization of stem cell derived cardiomyocytes Genomic, protein, metabolic, electrophysiological III. Methods for interrogating cardiomyocyte function Electrophysiology, Ca 2+ handling, Contractility Direct and Indirect (screening) methodologies IV. Case studies Retrospective Analysis V. Summary Mechanistic and Phenotypic Approaches

22 The Cardiac Action Potential Schram et al. Circ Res 2002 Fares and Howlett Proc. Aus. Physi. Soc. (2009) Basic functional electrical unit Drives contraction by initiating increases in intracellular Ca 2+ levels How are these processes interrogated in-vitro?

23 23 ipsc Cardiomyocytes Safety Pharmacology - APD Assay I Na Block Decreased dv/dt I Kr Block prolonged APD EAD Generation % of control Dose Peak MDP APD10 APD50 APD90 dv/dt I Ca Block shortened APD TTX N=5 E4031 N=5 Nifedipine N=5 1μM 103.8± ± ± ± ± ±7.4 3μM 100.0± ± ± ± ± ±11.2* 10μM 99.0± ± ± ± ± ±1.8* 30μM 99.1± ± ± ± ± ±2.0* 3nM 99.0± ± ± ± ± ±4.4 10nM 100.5± ± ± ± ± ±5.9 30nM 99.3± ± ± ±3.7* 140.3±7.6* 90.1± nM 101.1± ± ± ±3.9* ±13.6* 69.4±17.1 3nM 89.5± ± ±7.1* 84.6±2.4* 89.4±1.0* 83.9± nM 82.2± ± ±12.3* 70.3±6.1* 78.4±4.4* 91.3±4.4 30nM 87.2± ± ±7.6* 65.7±3.0* 74.0±2.3* 84.8± nM 73.6± ± ±11.1* 45.4±4.5* 58.2±5.4* 87.7±8.6 ipsc-cardiomyocytes show expected pharmacology Ma, et al, Am. J. Physiol., 2011

24 hesc Cardiomyocyte AP Pharmacology Arrhythmic Triggers Response Across Ion Channel Families Quantifiable Responses hesc-cardiomyocytes show expected pharmacology Peng et al.,

25 Stem cell Cardiomyocytes recapitulate pro-arrhythmia triggers EAD amplitude varies inversely with take off potential Adult Canine Purkinje Fiber APs EAD Take off potential ipsc CM APs Bay k ±0.26 mv/mv -2.28±0.11 mv/mv January et al, Circ Res, 65:570+, 1988 Ma et al, AJP:H&C, 301:H2006+,

26 msec mv mv mv Human ipsc Cardiomyocytes Organotypic Recordings MultiElectrode Array (MEA) Technique Waveforms 1 64 wells at a time 100 uv 500 ms 100 Nifedipine E Chromanol 293B Field Potential Overlays sfapdc *** *** control 10 nm 30 nm *** 300 * * control 3 nm 10 nm 30 nm 100 nm ** 200 control 10 mm 30 mm Nifedipine (nm) E-4031 (nm) Chromanol 293B (mm) Compounds induce expected effects 26

27 Human ipsc Cardiomyocytes Multi-electrode Array Measuring Conduction Velocity ipsc Human Cardiomyocyte Field Potentials 48-well MEA plate ipsc Human Cardiomyocyte FPD Initiation depends on well position within the plate ms ipsc Conduction velocity can be measured 1 0 Drug-induced effects on conduction velocity can be measured Organotypic preparations provide additional/alternative endpoints 27

28 Beats/min Beats/min RFU ipsc Cardiomyocytes Utility Ca 2+ Measurements Calcium 5 Assay Kit (Molecular Devices) Fluorescence intensity of Ca 2+ transients from autonomously beating cells. The entire experiment was finished in 100 seconds Beat rates before and after addition of 0.3 mm epinephrine Add compound Time, sec w format Concentration, um 4-P Fit: y = (A - D)/( 1 + (x/c)^b ) + D: A B C D R^2 iso (Isoproterenol: Concentration vs MeanValue) doxa (doxazosin: Concentration vs MeanValue) vera (verapamil: Concentration vs MeanValue) epi (Epinephrine: Concentration vs MeanValue) w format Concentration, um Concentration, um Positive Chronotropes: Isoproterenol Epinephrine Dopamine 1e Negative Chronotropes: 4-P Fit: y = (A - D)/( 1 + (x/c)^b ) + D: A B C D R^2 10 Plot#1 (Astemisole: Concentration vs MeanValue) e Plot#5 (TTX: Concentration vs MeanValue) Plot#4 (Cisarpide: Concentration vs MeanValue) Plot#2 (Pimoside: Concentration vs MeanValue) e Plot#3 (Terfenadine: Concentration vs MeanValue) Weighting: Fixed 1e P Fit: y = (A - D)/( 1 + (x/c)^b ) + D: A B C D R^2 Plot#5 (Doxazosin: Concentration vs MeanValue) e e Isoproterenol (Isoproterenol: Concentration vs Me Plot#3 (Epinephrine: Concentration vs MeanValue) Plot#2 (Propanolol: Concentration vs MeanValue) verapamil (Verapamil: Concentration vs MeanVal Doxasozine Verapamil Propranolol Modified from Sirenko et al.,

29 RFU 29 ipsc Cardiomyocytes Utility Ca 2+ Imaging Calcium 5 Assay Kit (Molecular Devices) Fluorescence intensity of Ca 2+ transients from autonomously beating cells. The entire experiment was finished in 100 seconds Beat rates before and after addition of 0.3 mm epinephrine Add compound Time, sec New algorithms are being developed for analysis Calcium 5 Response on FLIPR: Epinephrine High Dose (C06) Rise Time Peak Width (FWHM) Decay Time Amplitude & Time Time (sec)

30 30 Human ipsc Cardiomyocytes Functional Utility - Contractility Contraction Determined by edge detection Sunny Z. Sun Ca 2+ Transients Puppala et al, 2012

31 Human ipsc Cardiomyocytes Relevant Mitochondrial Toxicity Screens Assess viability while inhibiting mitochondrial function (rotenone, antimycin, and oligomycin) Media matters! Context inappropriate media can mask outcomes Decreased viability is masked by glucose Adapted from Rana et al.,

32 32 32 Presentation Outline I. Safety Pharmacology and in-vitro models Need for human cardiac model Potential for stem cell technology to provide a solution II. Functional characterization of stem cell derived cardiomyocytes Genomic, protein, metabolic, electrophysiological III. Methods for interrogating cardiomyocyte function Electrophysiology, Ca 2+ handling, Contractility Direct and Indirect (screening) methodologies IV. Case studies Retrospective Analysis V. Summary Mechanistic and Phenotypic Approaches

33 RFU 33 ipsc Cardiomyocytes Screening Ca 2+ Imaging reflects electrical activity Calcium 5 Assay Kit (Molecular Devices) Fluorescence intensity of Ca 2+ transients from autonomously beating cells. The entire experiment was finished in 100 seconds Beat rates before and after addition of 0.3 mm epinephrine Add compound Time, sec Electrical activity at the membrane is reflected in the Ca 2+ transient Calcium 5 Response on FLIPR: Epinephrine High Dose (C06) Calcium 5 Dye Response on FLIPR - Cisapride High Does (I12) Width ~ 1 sec Width ~ 8 sec Time (sec) Time (ms)

34 Relative Light Units Peak Count (120 sec) Relative Light Units Peak Count (120 sec) 34 ipsc-cardiomyocytes Screening FLIPR Tetra System - Quantitation No Treatment K7 - Group:Cisapride_9 - Concentration: mm Cisapride I7 - Group:Cisapride_7 - Concentration: pride ntration: Time (Seconds) Cisapride 15 min post addition EC/IC50 = Time (Seconds) Intracellular Ca 2+ oscillations can be used to measure membrane ion channel activity In well plates [Cisapride] (um)

35 Human ipsc Cardiomyocytes Label-Free Monitoring xcelligence RTCA Cardio Cell Index (log) Gold electrode Increasing Impedance 10 1 Seconds Slide modified from K. Kolaja 96-well E-eplate Time (hour) A surrogate measurement platform for electrical activity, Ca 2+ handling, contractility

36 ipsc-cardiomyocytes - Utility Label-Free Measurements Arrhythmogenesis Arrhythmia Screening in 96-wells More predictive than current models Identifies False Negatives identifies False Positives Guo et al., PPS - Predicted ProArhythmia Score 36

37 Presentation Outline I. Safety Pharmacology and in-vitro models Need for human cardiac model Potential for stem cell technology to provide a solution II. Functional characterization of stem cell derived cardiomyocytes Genomic, protein, metabolic, electrophysiological III. Methods for interrogating cardiomyocyte function Electrophysiology, Ca 2+ handling, Contractility Direct and Indirect (screening) methodologies IV. Case studies Retrospective Analysis V. Summary Mechanistic and Phenotypic Approaches

38 Example 1 38

39 Example 1 39

40 Example 2 40

41 Example 2 41

42 42 42 Presentation Outline I. Safety Pharmacology and in-vitro models Need for human cardiac model Potential for stem cell technology to provide a solution II. Functional characterization of stem cell derived cardiomyocytes Genomic, protein, metabolic, electrophysiological III. Methods for interrogating cardiomyocyte function Electrophysiology, Ca2+ handling, Contractility Direct and Indirect (screening) methodologies IV. Case studies Retrospective Analysis V. Summary Mechanistic and Phenotypic Approaches

43 Mechanistic Approach Functional Endpoints Example subset Process Cell Biology Wetware Endpoint Platform Electrical activity Ion channels GPCRs AP perturbation Chronotropic changes Patch Clamp, MEA, FliPR, HCI, Label-free Ca 2+ handling RYR2 SERCA Release and uptake FliPR, HCI, labelfree Contractility Contractile filaments Inotropic changes HCI, Ionoptix, label-free Bioenergetics Fatty Acid Oxidation Lipid Metabolism Mitochondrial function ATP production Oxygen consumption, mitochondrial dyes

44 Mechanistic Approach Functional Endpoints Example subset Process Cell Biology Wetware Endpoint Platform Electrical activity Ion channels GPCRs AP perturbation Chronotropic changes Patch Clamp, MEA, FliPR, HCI, Label-free Ca 2+ handling RYR2 SERCA Release and uptake FliPR, HCI, labelfree Contractility Contractile filaments Inotropic changes HCI, Ionoptix, label-free Bioenergetics Fatty Acid Oxidation Lipid Metabolism Mitochondrial function ATP production Oxygen consumption, mitochondrial dyes adverse signal Phenotypic Approach Consistent output Surrogate measurements

45 45 Summary Human Stem Cell Derived Cardiomyocytes Human cardiomyocytes Provide an in-vitro recapitulation of native cellular biology and physiology Can be used across a variety of interrogation platforms Have, in some cases, shown greater utility than current models Can be used for phenotypic and mechanistic screening investigations

46 Thank-you